The cellular mechanisms by which nervous systems evolve to match evolutionary changes occurring in the rest of the body remain largely unexplored. In a distal visual neuropil of a previously unexamined ancient dipteran family, Stratiomyidae, homologues of all of the periodic neurons known already from more recent Diptera can be recognized, occupying the same locations within the unit structure. This points to extreme developmental stasis for more than 200 million years, conserving both cell identity and position. The arborizations that some neurons make also have remained conservative, but others show marked differences between families in both size and branching patterns. At the electron-microscopical level, extensive differences in synaptic connectivity are found, some sufficient to radically redefine the systems roles of particular neurons. The findings bear out an earlier prediction that changes in the connectivity matrix linking conserved neurons may have been a major factor in implementing evolutionary change in the nervous system.

This study investigated the effects of repeated administrations of nicotine (0.7 mg/kg) on dendritic morphology in the nucleus accumbens (NAcc), prefrontal cortex (Cg 3), and parietal cortex (Par 1). Animals were habituated for 3 days to a locomotor box, and after habituation, every second day for 5 weeks rats were placed into the locomotor chamber immediately after a subcutaneous injection of nicotine or saline. Rats demonstrated tolerance to an initial hypoactive response after each nicotine injection, and this was followed by an increase in activity after each injection (behavioral sensitization). This increase in activity was still present on a nicotine challenge after a 2-week abstinence period. One week after the nicotine challenge day, all rats were perfused and brains were removed. These brains we stained using Golgi-Cox procedures, and dendrites from the nucleus accumbens (N Acc), medial frontal cortex (Cg 3) and parietal cortex (Par 1) were analyzed using the camera lucida procedure. Results showed that rats receiving nicotine demonstrated an increase in dendritic length and spine density relative to controls in the NAcc and Cg3 brain areas, but not Par 1. The increase observed in the NAcc was significantly greater than what has been found with amphetamine or cocaine, and possible underlying mechanisms were discussed.

The use of peptides in drug development has been hampered by their poor pharmaceutical properties, most notably their inability to reliably permeate biological membranes and lack of targeting. To overcome these disadvantages, the AMino acid Intracellular Delivery SysTem (AMIDST) was developed. This modular peptide-based delivery system confers cellular permeability and organelle-specific targeting for therapeutic peptides. As demonstrated in this study, the delivery of a HER2-binding peptide to the secretory organelles of breast cancer cells resulted in intracellular sequestration, a reduction in downstream signalling, and reduced viability compared to the delivery of a control peptide. Given its modular design and ease of production, AMIDST has the potential to enhance the use of peptides as therapeutic agents.

Cerebral palsy (CP) as a neurodevelopmental disorder that affects individuals’ movement, posture, and balance and occurs in every 2-3 out of 1,000 live births. Symptoms of CP can include seizures, hydrocephalus, impairment of the limbs, and learning disabilities. External contributors to CP are well known, but there are 80% of CP cases are idiopathic and in which no brain injury is reported. Recently, several genetic studies have shown that deleterious de novo mutations in CP patients may be implicated in CP pathogenesis. One such potentially deleterious de novo mutation of RhoB was identified in two CP patients. RhoB encodes for RHOB protein, a Rho GTPase that regulates the actin cytoskeleton. Biochemical and structural analyses of RhoB (S73F) protein suggested that the RhoB mutation generates a hyperactive form of RhoB. However, how the RhoB (S73F) protein may interfere with brain development and can contribute to CP is unknown. To determine whether RhoB (S73F) expression affects cortical development, we used in utero electroporations in mice to study the effect of RhoB (S73F) expression on cellular morphology, polarity, migration, and Golgi localization in the embryonic mouse model at E15.5 and P0, comparing it to a RhoB overexpression model as well as control. To address changes in cell morphology, we examined the cell size, shape, and volume of RhoB expressing cells using Imaris software. We show that RhoB overexpression and RhoB (S73F) expression cause detrimental changes in cell shape, polarity, and neuritogenesis. Furthermore, RhoB (S73F) expressing cells migrate less compared to RhoB overexpressing cells and control. Interestingly, we found that RhoB (S73F) expressing cells that did not migrate away from the ventricular surface still became neurons. To determine the effect of RhoB (S73F) expression on the subcellular environment, we examined the localization of the Golgi apparatus, and found the Golgi to be mislocalized and fragmented when RhoB (S73F) was expressed. Overall, this study shows that overexpression of RhoB is sufficient to cause changes in cell morphology, polarity, migration, and subcellular localization of the Golgi. Importantly, expression of RhoB (S73F) is distinct and unique from RhoB overexpression, causing more severe changes in cell size, shape, polarity, cell process number, and Golgi localization that result in failed neuronal migration. This data suggests the potential for genetic mutations to enact changes within the structure and function of cortical cells, which may contribute to the pathogenesis of CP. / Cell Biology

Among Human Immunodeficiency viruses (HIV), type-1 (HIV-1) is the most common worldwide and has the highest virulence and infectivity. Though the virus only infects a few cell types, the infection affects almost every organ system causing multiple comorbidities. One of the comorbidities is HIV-associated Neurocognitive Disorders (HAND). Interorganelle communication regulates many cellular functions including calcium exchange, lipid exchange, intracellular trafficking, and mitochondrial biogenesis. Interestingly, all these processes have been implicated in HAND suggesting that dysregulation of interorganelle communication plays a role in the progression of HAND. Using neuronal cell cultures, I show that mitochondrial-associated ER membranes (MAM)-associated protein and MAM-tethering protein expression and interactions are altered in the presence of the HIV-1 protein Tat. I also show, PTPIP51 and VAPB, two MAM-tethering proteins, expression is altered in the MAM fraction but not the whole cell fraction, indicating a localization problem. Phosphorylation of PTPIP51 has been shown to regulate the subcellular localization and I show that tyrosine phosphorylation is upregulated with Tat. In addition, I show that PTPIP51 binding with VAPB can be rescued with the addition of kinase inhibitors blocking PTPIP51 phosphorylation suggesting that Tat is altering the phosphorylation of PTPIP51 affecting its subcellular localization and binding to VAPB. Furthermore, I show that ER-Golgi communication is altered in the presence of Tat where there is an increase in the interactions between YIF1A and VAPB, two ER-Golgi tethering proteins. The altered iv interactions between MAM and ER-Golgi tethering proteins in the presence of Tat lead to the disruption of cellular pathways associated with dysfunctional interorganelle communication that can lead to neuronal dysfunction and can contribute to HAND. / Biomedical Sciences

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Transport of different organelles along the Microtubule cytoskeleton is carried out mainly by motor proteins Dynein and Kinesin. The tubulin monomers in Microtubules are organized in such a way that the generate polarity (a minus and a plus end) that is recognized by Motor proteins. Dynein usually acts with a binding partner, Dynactin, and is in charge of moving cargoes to the minus end of microtubules (mainly towards the center of the cell). There are different kinesins, the most studied is Kinesin-1, which moves cargoes towards the plus end of microtubules. In order to fulfil their function Motors usually bind to their cargoes indirectly through adaptor proteins. Chapter 1 explains the general concepts related to a group of Adaptors that recognize the small GTP-ases, Rabs, in cargoes that need to be transported under certain physiological circumstances and help recruiting the Dynein/Dynactin complexes to them so they can move in the minus end direction. This family of Adaptors is called Rab-Dynein-Dynactin (RDD) adaptors and in this project I focused on two of them: BicD2 and RILP. In chapter 2, I will focus on BicD2 and its role in Golgi morphology. BicD2 is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab6-positive vesicles. Some mutations in BicD2 have been associated to Golgi apparatus morphology disruption, but the mechanism is unclear. It has been suggested that mutated BicD2 abnormally binds Dynein/Dynactin, sequestering this motor complex, producing Golgi disruption indirectly since this organelle depends heavily on minus-directed transport to maintain its localization and structure. I test this hypothesis and conclude that even when most pathological mutations disrupt the Golgi, a Dynein/Dynactin-mediated mechanisms is probably true only to some of them, proposing alternatives mechanisms such as Rab6 abnormal accumulation and non-Golgi related mechanisms of pathogenesis. In chapter 3, I will focus on RILP and its role in autophagosome movement. RILP is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab7-positive vesicles such as Lysosomes. During autophagy, autophagosomes (which are LC3-positive) are formed mainly in the ER and mature to finally fuse with the Late Endosomes or Lysosomes (both acidic) in the center of the cell. It has been described by our lab that RILP can transport LC3-vesicles in axons. Nevertheless, these vesicles are acidic, which suggest these LC3-vesicles are already fused with either Lysosomes or Late endosomes. I will work under the Hypothesis that RILP can move autophagosomes in early stages (before fusion with Lysosomes or Late endosomes) in non-neuronal cells. I show that RILP can move autophagosomes to the center and FYCO1 (a Kinesin-1 adaptor) can move them to the periphery. RILP-mediated movement of autophagosomes depends on Rab7 activation status and seems to be controlled by PKA. I proposed a phosphorylation in Rab7 as a control mechanism. Finally, the discovery of 3 LC3 interacting regions (LIRs) in the RILP molecule is discussed and their contribution to autophagosome movement is analyzed. My results highlight the relevance of RDD proteins in physiological and pathological context.

The research presented in this dissertation consists of two primary parts. The initial focus centered on understanding the distribution of Golgi resident glycosyltransferases between the ER and Golgi at steady-state. Retrograde trafficking of these Golgi proteins has been demonstrated experimentally mandating the existence of a dynamic equilibrium between the Golgi apparatus and ER. Our published studies also included the development of a quantitative method for analysis of data collected using fluorescent microscopy. The second part of this dissertation presents results pertaining to the quantification of a unique Golgi resident protein that cycles in the late endosome bypass pathway. Using the published method of analysis and techniques developed during the initial project, the anterograde and retrograde transport kinetics of this Golgi protein were determined and used to develop a compartmental model for pH sensitive trafficking in the bypass pathway. The spatial Golgi distribution of the protein during retrograde transport to the Golgi following endosomal exit was also investigated. This research lies at the interface of experimental cell biology and quantitative computational analysis. These experiments combined more traditional experimental biological approaches with more recent computational approaches to understanding cellular mechanisms. Additionally, development of a quantitative method of analysis validated the use of fluorescent microscopy as a quantitative tool for studying intracellular proteins. / Ph. D.

À mon arrivée dans le laboratoire, les travaux réalisés indiquaient que le trafic des endosomes de recyclage (ERs) vers le Golgi était régulé par les kinases de la famille Src (KFS) et contribuait au remodelage de l’actine induit par différents stress (E4orf4, staurosporine). Or les KFS coordonnent la dynamique de l’actine et le trafic membranaire, mais les mécanismes impliqués demeurent peu caractérisés. Des évidences récentes indiquaient aussi que le trafic endosomal contribuait à la transformation cellulaire induite par des formes oncogéniques des KFS, laquelle est associée à l’assemblage de structures invasives riches en actine (podosomes). En se basant sur ces données, notre hypothèse de recherche était que le transport des ERs vers le Golgi sous le contrôle du rétromère, un régulateur majeur des voies de transport rétrograde, pourrait contribuer à la formation des podosomes lors de la transformation cellulaire induite par v-Src. Les objectifs du travail présenté dans ce mémoire étaient de caractériser les changements précoces dans le trafic endosomal dépendant du rétromère suivant l’activation de v-Src et d’adresser leur contribution dans la formation des podosomes. Mes travaux ont tiré profit de l’utilisation d’un modèle permettant l’activation rapide d’un mutant thermosensible de v-Src dans des cellules épithéliales MDCK. J’ai ainsi pu observer dans ce modèle une polarisation précoce des ERs autour des podosomes en formation. Une augmentation dans le nombre et la morphologie des endosomes rétromère-dépendants (Vps26) a été mesurée et coïncidait avec un transport accru de cargos dépendants du rétromère vers le Golgi. Notamment, l’inhibition de Rab7, un régulateur du rétromère, interfère avec la formation des endosomes Vps26-positifs et des podosomes. En conclusion, mes résultats suggèrent que v-Src active les voies de transport dépendantes du rétromère pour mobiliser des protéines de signalisation et/ou des lipides bioactifs nécessaires au remodelage polarisé de l’actine et à la formation des structures invasives.

Yes / Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell.